The present invention relates to a new method for the amplification and stabilization of the copy number of chosen genes in a microorganism.
The invention is illustrated by means of examples given hereinafter in which the sequence coding for an enzyme, beta-galactosidase, and the genes coding for three enzymes involved in the biosynthesis of L-threonine are transferred inside the chromosome of a microorganism.
These genes have been shown to be stably fixed in the chromosome. The resulting strains are therefore better producers of, in the first instance, the corresponding enzymes or, secondly, of L-threonine amino acids, than are the parental strains.
The bacteria are capable of synthesizing useful active principles such as enzymes and amino acids. The production by fermentation involves complex biochemical procedures controlled by multiple genes and gene products. The products of the fermentation are obtained in relatively small quantities. In order to improve this production by fermentation, it would be useful to increase the number of copies of the genes involved, in a manner such that the number of these copies could be controlled and stabilized in the chromosome or the episomes of the microorganism.
Several methods have been described in the scientific literature in which the copy number of a gene can be amplified in a microorganism, in particular by means of circular plasmid vectors. However, the number of copies obtained by these methods is extremely variable and depends on the particular vector, the length of the chosen gene and the particular physiological conditions of the microorganism. None of these methods are concerned with the amplification of the gene to a chosen level, avoiding an unnecessary excess of copies, and this with a stable fixation of these copies in the chromosome.
Various documents suggest the use of transposons as vectors in bacteria or other organisms, in particular:
U.S. Pat. No. 4,670,388, PA1 EP-A-0,200,708 PA1 EP-A-0,091,723 PA1 GENE, vol. 42 (1986), pages 185-192 PA1 THE EMBO JOURNAL, vol 4, No. 4, 1985, pages 891-898, and PA1 BIOTECHNOLOGY, vol. 4, No. 5, May 1986, pages 446-449, New York, U.S. PA1 a) the said chosen gene or the chosen DNA sequence is cloned inside a defective transposon outside the essential parts of the transposon, PA1 b) the said transposon is integrated in the DNA sequence inside the bacterium. PA1 a) the said chosen gene or the chosen DNA sequence is cloned inside a transposon outside the essential parts of the transposon, the said transposon being defective, PA1 b) the said transposon is integrated in the DNA sequence such as the chromosome or the episome of the said bacterium, PA1 c) the said defective transposon is complemented so as to be able to transpose several times and the complementation is then stopped after a specific number of transpositions.
However, none of these documents describes a process by which it is possible to obtain a stable integration of a specific DNA sequence with a defined copy number.